Figure 1.

Strategy of the three-plasmid integron selection system. The system consists of three plasmids: p1 expresses the class 1 integron-integrase gene, intI1 (white arrow); p2 is a self-transmissible recipient plasmid that contains a class 1 integron (white arrows); p3 is the substrate plasmid into which genomic DNA is cloned. A genomic library is constructed in p3 (middle panel), some of which will contain integron gene cassettes (p3c; white arrows, orfs; black arrowheads, attC sites). The rest will contain other chromosomal DNA (p3o; black arrows, orfs). A specialized E. coli strain that allows for easy antibiotic-independent counter-selection by the simple omission of a specific nutrient from the media is used as the host strain. p2 is transferred by conjugation (top left panel) to the E. coli strain containing the library. The transconjugants are pooled and transformed with p1 (bottom left panel) to complete assembly of the system (top right panel). Integrase-mediated recombination between the attI1 site of p2 and target attC sites on p3c fuses these plasmids together, while p3o plasmids are not recombined. The fusions are selected following conjugation to a second E. coli strain and plating on the appropriate selective media that lacks the specific nutrient (bottom right panel).